Construction of pLLO vector encoding truncated form of Listeriolysin O as molecular adjuvant for DNA vaccine studies
Archives of Medical Laboratory Sciences,
Vol. 2 No. 3 (2016),
23 November 2016
https://doi.org/10.22037/amls.v2i3.15464
Abstract
Background: The major problem of DNA vaccine is less immunogenicity of them verses other killed or live whole organism vaccines therefore adjuvants for use in this kind vaccines is very necessary. Genetic adjuvants with bacterial sources are an appropriate approach to modulate immune responses to DNA vaccines. Listeria Monocytogenes proteins such as Listeriolysin O (LLO) with CD4 and CD8 epitopes can be as an adjuvant to initiate both innate and adaptive immune responses if the protein cytotoxicity can be eliminated. Herein we constructed a truncated LLO plasmid as genetic adjuvant and tested it in combination with a DNA construct as a model vaccine.
Materials and Methods: About 1340bp of the 5' end of whole LLO gene was amplified by PCR on DNA purified from Listeria Monocytogenes. Sequential sub cloning of truncated LLO into the Xho I/EcoRV sites of pcDNA3.1 plasmid, downstream of CMV promoter was done. pLLO plasmid was transfected to HEK293T cell line by lipofection method. LLO protein expression from transiently transfected 293T cell lysates was confirmed by western blotting. Then the adjuvant activity of LLO in BALB/c mice model was analyzed using proliferation test.
Results: Double digestion of pLLO plasmid with the enzymes that were applied for cloning led to the isolation of two fragments with expected sizes. The final plasmid was also confirmed following sequencing reactions. Moreover, expression of LLO was evidenced in transfected 293T cells, compared to non-transfected controls. In vivo study was shown, high significant proliferative responses in LLO co-immunization pattern.
Conclusion: In the DNA vaccine study, LLO co-administration plasmid could be a suitable genetic adjuvant to enhance cellular immune response of vaccine.
- DNA vaccine
- molecular adjuvant
- Listeria Monocytogenes
- listeriolysin O
How to Cite
References
Petrovsky N, Aguilar JC. Vaccine adjuvants: current state and future trends. Immunology and cell biology. 2004;82(5):488-96.
Scheerlinck J-PY. Genetic adjuvants for DNA vaccines. Vaccine. 2001;19(17):2647-56.
Schnupf P, Portnoy DA. Listeriolysin O: a phagosome-specific lysin. Microbes and Infection. 2007;9(10):1176-87.
Gilbert RJ. Cholesterol-dependent cytolysins. Proteins Membrane Binding and Pore Formation: Springer; 2010. p. 56-66.
Hernández-Flores K, Vivanco-Cid H. Biological effects of listeriolysin O: implications for vaccination. BioMed research international. 2015;2015.
Greten FR, Eckmann L, Greten TF, Park JM, Li Z-W, Egan LJ, et al. IKKβ links inflammation and tumorigenesis in a mouse model of colitis-associated cancer. Cell. 2004;118(3):285-96.
Darji A, Chakraborty T, Wehland J, Weiss S. Listeriolysin generates a route for the presentation of exogenous antigens by major histocompatibility complex class I. European journal of immunology. 1995;25(10):2967-71.
Neeson P, Pan ZK, Paterson Y. Listeriolysin O is an improved protein carrier for lymphoma immunoglobulin idiotype and provides systemic protection against 38C13 lymphoma. Cancer Immunology, Immunotherapy. 2008;57(4):493-505.
Michel E, Reich K, Favier R, Berche P, Cossart P. Attenuated mutants of the intracellular bacterium Listeria monocytogenes obtained by single amino acid substitutions in listeriolysin O. Molecular microbiology. 1990;4(12):2167-78.
Pouriayevali M-H, Bamdad T, Aghasadeghi M-R, Sadat SM, Sabahi F. Construction and Immunogenicity Analysis of Hepatitis C Virus (HCV) Truncated Non-Structural Protein 3 (NS3) Plasmid Vaccine. Jundishapur journal of microbiology. 2016;9(3).
Jin H, Li Y, Ma Z, Zhang F, Xie Q, Gu D, et al. Effect of chemical adjuvants on DNA vaccination. Vaccine. 2004;22(21):2925-35.
Zavvar M, Moshiri A, Motevali F, Pouriayevali M, Bahramali G. Construction of pIRES2 vector encoding truncated NS3 of HCV and IL-18 for DNA vaccine studies. Vaccine Research. 2015;2(3):24-8.
Carrero JA, Vivanco-Cid H, Unanue ER. Listeriolysin O is strongly immunogenic independently of its cytotoxic activity. PLoS One. 2012;7(3):e32310.
Sun R, Liu Y. Listeriolysin O as a strong immunogenic molecule for the development of new anti-tumor vaccines. Human vaccines & immunotherapeutics. 2013;9(5):1058-68.
Carrero JA, Calderon B, Unanue ER. Listeriolysin O from Listeria monocytogenes is a lymphocyte apoptogenic molecule. The Journal of immunology. 2004;172(8):4866-74.
Beattie I, Swaminathan B, Ziegler H. Cloning and characterization of T-cell-reactive protein antigens from Listeria monocytogenes. Infection and immunity. 1990;58(9):2792-803.
Guzmán CA, Domann E, Ronde M, Bruder D, Darji A, Weiss S, et al. Apoptosis of mouse dendritic cells is triggered by listeriolysin, the major virulence determinant of Listeria monocytogenes. Molecular microbiology. 1996;20(1):119-26.
Köster S, van Pee K, Hudel M, Leustik M, Rhinow D, Kühlbrandt W, et al. Crystal structure of listeriolysin O reveals molecular details of oligomerization and pore formation. Nature communications. 2014;5.
Rosado CJ, Kondos S, Bull TE, Kuiper MJ, Law RH, Buckle AM, et al. The MACPF/CDC family of pore‐forming toxins. Cellular microbiology. 2008;10(9):1765-74.
Kohda C, Kawamura I, Baba H, Nomura T, Ito Y, Kimoto T, et al. Dissociated linkage of cytokine-inducing activity and cytotoxicity to different domains of listeriolysin O from Listeria monocytogenes. Infection and immunity. 2002;70(3):1334-41.
- Abstract Viewed: 533 times
- PDF Downloaded: 390 times